FIG 3.

Dynasore blocks HSV-1 and HSV-2 entry into human neuronal cells. (A) SK-N-SH cells were pretreated with dynasore, heparin, or a control buffer and were then exposed to the virus at 37°C for 1 h. Western blots of nuclear extracts were probed with antibodies to VP16, a viral tegument protein that is transported to the nucleus within the first hour of viral entry, and β-actin (as a control). (B) SK-N-SH cells were pretreated with the drugs for 1 h at 37°C, then cooled to 4°C, a temperature permissive for viral binding but not entry, and finally exposed to HSV-2 at an MOI of 0.1 or 1 PFU/cell for 4 h. The cells were then washed, and cell lysates were prepared and were analyzed for the presence of bound viral particles by immunoblotting for the viral envelope glycoprotein D (gD). (C) Dynasore- or control buffer-treated SK-N-SH or primary fetal neuronal cells were synchronously infected with HSV-1(KVP26GFP) and were then fixed and stained with DAPI (blue). Confocal images were obtained 1 h after incubation at 37°C. Images of representative cells are shown. (D) GFP-positive nuclei were counted in five independent fields (100 cells), and percentages are shown. Asterisks indicate P values of <0.01 (by t test). (E) SK-N-SH cells were infected with HSV-1(KVP26GFP) and, after 1 h, were washed with a low-pH citrate buffer to inactivate extracellular virus. Then dynasore or a control buffer was added for an additional 1 h of incubation. Cells were then fixed and stained with DAPI (blue), and extended-focus confocal images were obtained. Two representative fields are shown for each condition. (F) Vaginal epithelial cells (VK2/E6/E7) were pretreated with 60 μM dynasore or a control buffer and were subsequently exposed to Alexa Fluor 555-conjugated transferrin (red) or a buffer (no transferrin) as described in Materials and Methods. Representative extended-focus images are shown. The plasma membranes were stained green with the lipophilic tracer DiO; cells were fixed; and nuclei were stained blue with DAPI. Images and blots are representative of at least two independent experiments.